Gene therapy is a rapidly emerging field that aims to treat acquired diseases such as cancer, diabetes and AIDS as well as inherent metabolic abnormality. Among several gene delivery vector systems currently used in gene therapy, the retrovirus vector system has advantages over others using adenovirus, liposome, electroporation and gene gun. For example, the retrovirus vector system has the ability to permanently integrate into host cells that allows stable expression of transduced cells. However, all of these systems have limitation in gene delivery to a specific target cell.
Several methods have been developed for specifically introducing a therapeutic gene into target cells using a retrovirus, e.g.; a method for linking a sugar molecule to an envelop glycoprotein of retrovirus by a chemical method so that the sugar-coupled envelope glycoprotein binds to an asialoglycoprotein receptor of target cells (Neda, H. et al., J. Biol. Chem. 266: 14143-14146, 1991); a method of using a coupling antibody bridge capable of binding to both of an envelop glycoprotein of retrovirus and a target cell's receptor or surface antigen (Goud, B. et al., Virology 163: 251-254, 1988; Roux, P. et al., Proc. Natl. Acad. Sci. U.S.A. 86: 9079-9083, 1989; Etienne-Julan, M. et al., J. Gen. Virol. 73: 3251-3255, 1992); a method for coupling a single-chain antibody to an envelope glycoprotein of retrovirus via genetic engineering and infecting target cells with the retrovirus (Russell, S. J. et al., Nucleic Acids Res. 21: 1081-1085, 1993; Somia, N. V. et al., Proc. Natl. Acad. Sci. U.S.A. 92: 7570-7574, 1995; Ager, S. et al., Hum. Gene Ther. 7: 2157-2164, 1996; Marin, M. et al., J. Virol. 70: 2957-2962, 1996; Schnierle, B. S. et al., Gene Ther. 3: 334-342, 1996); and a method for coupling a peptide ligand to the envelop glycoprotein of retrovirus via genetic engineering and infecting target cells with the retrovirus (Kasahara, N. et al., Science 266: 1373-1376, 1994; Cosset, F. L. et al., J. Virol. 69: 6314-6322, 1995; Schnierle, B. S., and Groner, B., Gene Ther. 3: 1069-1073, 1996). However, these methods show very low transduction efficiency.
There have also been developed methods using an avian or a murine retrovirus to introduce a therapeutic gene into human target cells (Chu, T-H. T., and R. Domburg, J. Virol. 69: 2659-2663, 1995; Chu, T-H. T. et al., Gene Ther. 1: 292-299, 1994; Valsesia-Wittmann, S. et al., J. Virol. 68: 4609-4619, 1994; Cosset, F-L. et al., J. Virol. 69: 6314-6322, 1995; Han, X. et al., Proc. Natl. Acad. Sci. USA 92: 9747-9751, 1995; Kasahara, N. et al., Science 266: 1373-1376, 1994; Somia, N. V., et al., Proc. Natl. Acad. Sci. USA 92: 7570-7574, 1995; HAN, J. Y., et al., J. Virol. 71, 8103-8108, 1997). The structure and function of a murine retrovirus have been widely studied, and, in particular, the tertiary structures of Mo-MuLV and Fr-MuLV envelope glycoproteins have been recently established by X-ray crystallography.
If the receptor or surface antigen of target cells is identified and specific, a method for coupling a single-chain antibody (ScFv) to an envelope glycoprotein of retrovirus via genetic engineering and infecting target cells with the retrovirus is very useful for the specific infection of target cells.
Single chain antibody (ScFv) is formed by joining the carboxy-terminal of VH and the amino-terminal of VL segments with a suitable synthetic amino acid linker. The antibody combining site is located at the Fv region of the molecule formed by the variable domains of heavy and light chains (VH and VL) (Poon et al., Molecular Immunology 39: 19-24, 2002; Fujiwara et al., Biochemistry 41: 12729-12738, 2002).
Recently, the pseudotyping of retrovirus has been studied to improve stability and transduction efficiency. For example, a gibbon ape leukemia virus (GaLV) Env-pseudotype retroviral vector showed 5 to 30 times higher infection efficiency than an amphotropic murine retroviral vector in several human cell lines (Kim et al., Proc. Amer. Assoc. Cancer Res. 38: 177, 1997). However, the limitation in specific targeting is remained to be solved.
Therefore, the present inventors have endeavored to meet the need of a gene delivery system using a retrovirus which shows higher viral titer and transduction efficiency, and develop a chimeric ligand in the form of a fusion polypeptide of the GaLV envelope glycoprotein and a single chain antibody derived from monoclonal antibody capable of specifically binding to a surface antigen of tumor associated glycoprotein 72 (Tag-72).